NADH Oxidation Research Articles

Functional Nanozymes Consisting of Co2+‐ZIF‐67 Metal‐Organic Framework Nanoparticles and Co2+‐ZIF‐67/Polyaniline Conjugates

AbstractCo2+‐ZIF‐67 metal‐organic framework nanoparticles (NMOFs), act as nanozymes catalyzing diverse processes, including peroxidase, oxidase, and catalase activities. Peroxidase activities are reflected by the nanozyme‐catalyzed H2O2 oxidation of dopamine to aminochrome, the H2O2 oxidation of NADH to NAD+, the H2O2‐catalyzed generation of chemiluminescence through oxidation of luminol, and the H2O2‐mediated oxidation of aniline to polyaniline. Oxidase activities of the nanozyme are demonstrated by the Co2+‐ZIF‐67 NMOFs catalyzed aerobic oxidation of dopamine to aminochrome and of NADH to NAD+, and catalase activities of the nanozyme are reflected by the catalytic decomposition of H2O2. Moreover, the Co2+‐ZIF‐67 catalyzed oxidation of aniline to polyaniline (PAN) by H2O2 is accompanied by the coating of NMOFs with PAN to yield a Co2+‐ZIF‐67/PAN hybrid material. Coating the particles in the presence of guest substrates leads to molecular imprinted PAN matrices revealing enhanced ZIF‐67 catalyzed transformations as compared to non‐imprinted PAN matrices. This is demonstrated by imprinting of 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid), ABTS2−, in the PAN coating and the 3.3‐fold enhanced catalyzed oxidation of ABTS2− by H2O2, as compared to the non‐imprinted PAN hybrid composite. Moreover, imprinting of L‐/D‐DOPA in the PAN coating of Co2+‐ZIF‐67 NMOFs leads to chiro‐selective H2O2‐guided oxidation of L‐/D‐DOPA to dopachrome by the NMOFs/PAN composite.

Co-cultivation of Saccharomyces cerevisiae strains combines advantages of different metabolic engineering strategies for improved ethanol yield

Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae. Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions. Functional expression of heterologous phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes enables yeast cells to couple a net oxidation of NADH to the conversion of glucose to ethanol. In another strategy, NADH-dependent reduction of exogenous acetate to ethanol is enabled by introduction of a heterologous acetylating acetaldehyde dehydrogenase (A-ALD). This study explores potential advantages of co-cultivating engineered PRK-RuBisCO-based and A-ALD-based strains in anaerobic bioreactor batch cultures. Co-cultivation of these strains, which in monocultures showed reduced glycerol yields and improved ethanol yields, strongly reduced the formation of acetaldehyde and acetate, two byproducts that were formed in anaerobic monocultures of a PRK-RuBisCO-based strain. In addition, co-cultures on medium with low acetate-to-glucose ratios that mimicked those in industrial feedstocks completely removed acetate from the medium. Kinetics of co-cultivation processes and glycerol production could be optimized by tuning the relative inoculum sizes of the two strains. Co-cultivation of a PRK-RuBisCO strain with a Δgpd1 Δgpd2 A-ALD strain, which was unable to grow in the absence of acetate and evolved for faster anaerobic growth in acetate-supplemented batch cultures, further reduced glycerol formation but led to extended fermentation times. These results demonstrate the potential of using defined consortia of engineered S. cerevisiae strains for high-yield, minimal-waste ethanol production.

Conformational coupling of redox-driven Na+-translocation in Vibrio cholerae NADH:quinone oxidoreductase

In the respiratory chain, NADH oxidation is coupled to ion translocation across the membrane to build up an electrochemical gradient. In the human pathogen Vibrio cholerae, the sodium-pumping NADH:quinone oxidoreductase (Na+-NQR) generates a sodium gradient by a so far unknown mechanism. Here we show that ion pumping in Na+-NQR is driven by large conformational changes coupling electron transfer to ion translocation. We have determined a series of cryo-EM and X-ray structures of the Na+-NQR that represent snapshots of the catalytic cycle. The six subunits NqrA, B, C, D, E, and F of Na+-NQR harbor a unique set of cofactors that shuttle the electrons from NADH twice across the membrane to quinone. The redox state of a unique intramembranous [2Fe-2S] cluster orchestrates the movements of subunit NqrC, which acts as an electron transfer switch. We propose that this switching movement controls the release of Na+ from a binding site localized in subunit NqrB.

NADH Photosynthesis System with Affordable Electron Supply and Inhibited NADH Oxidation

AbstractPhotosynthesis offers a green approach for the recycling of nicotinamide cofactors primarily NADH in bio‐redox reactions. Herein, we report an NADH photosynthesis system where the oxidation of biomass derivatives is designed as an electron supply module (ESM) to afford electrons and superoxide dismutase/catalase (SOD/CAT) cascade catalysis is designed as a reactive oxygen species (ROS) elimination module (REM) to inhibit NADH degradation. Glucose as the electron donor guarantees the reaction sustainability accompanied with oxidative products of gluconic acid and formic acid. Meanwhile, enzyme cascades of SOD/CAT greatly eliminate ROS, leading to a ≈2.00‐fold elevation of NADH yield (61.1 % vs. 30.7 %). The initial reaction rate and turnover frequency (TOF) increased by 2.50 times and 2.54 times, respectively, compared with those systems without REM. Our study establishes a novel and efficient platform for NADH photosynthesis coupled to biomass‐to‐chemical conversion.

NADH Photosynthesis System with Affordable Electron Supply and Inhibited NADH Oxidation.

Photosynthesis offers a green approach for the recycling of nicotinamide cofactors primarily NADH in bio-redox reactions. Herein, we report an NADH photosynthesis system where the biomass derivatives oxidation is designed as an electron supply module (ESM) to afford electrons and the superoxide dismutase/catalase (SOD/CAT) cascades catalysis is designed as reactive oxygen species (ROS) elimination module (REM) to inhibit NADH degradation. Glucose as the electron donor guarantees the reaction sustainability accompanied with oxidative products of gluconic acid and formic acid. Meanwhile, enzyme cascades of SOD/CAT greatly eliminate ROS, leading to a ~2.00-fold elevation of NADH yield (61.1% vs. 30.7%). The initial reaction rate and turnover frequency (TOF) increased by 2.50 times and 2.54 times, respectively, compared with those systems without REM. Our study establishes a novel and efficient platform for NADH photosynthesis coupled to biomass-to-chemical conversion.

Half-Sandwich Iridium(III), Rhodium(III), and Ruthenium(II) Complexes Chelating Hybrid sp2-N/sp3-N Donor Ligands to Achieve Improved Anticancer Selectivity.

The biological efficacy of half-sandwich platinum group organometallic complexes of the formula [(η5-Cpx)/(η6-arene)M(XY)Cl]0/+ (XY = bidentate ligands; Cpx = functionalized cyclopentadienyl; M = Ir, Rh, Ru, Os) has received considerable attention due to the significance of the metal center, chelating ligand, and Cpx/arene moieties in defining their anticancer potency and selectivity. With a facile access to the BIAN-derived imine-amine ligands using alkylaluminum as the reductant, we herein described the preparation and characterization of 16 half-sandwich Ir(III), Rh(III), and Ru(II) complexes chelating the hybrid sp2-N/sp3-N donor ligand. A nonplanar five-member metallacycle was confirmed by X-ray single-crystal structures of Ir1-Ir3, Ir7, Rh1, Ru1, and Ru4. The attempt to prepare imine-amido complexes using a base as the deprotonating agent led to the mixture of imine-amine complexes, within which the leaving group Cl- was displaced, and 16-electron imine-amido complexes without Cl-. The half-sandwich imine-amine complexes in this system underwent rapid hydrolysis in aqueous solution, exhibited weak photoluminescence, and showed the ability of binding to CT-DNA and BSA. The cytotoxicity of all imine-amine complexes against A549 lung cancer cell lines, HeLa cervical cancer cell lines, and 4T1 mouse breast cancer cells was determined by an MTT assay. The IC50 values of these complexes were in a range of 5.71-67.28 μM. Notably, most of these complexes displayed improved selectivity toward A549 cancer cells versus noncancerous BEAS-2B cells in comparison with the corresponding α-diimine complexes chelating the sp2-N/sp2-N donor ligand, which have been shown no selectivity in our previous report. The anticancer selectivity of these complexes appeared to be related to the redox-based mechanism including the catalytic oxidation of NADH to NAD+, reactive oxygen species (ROS) generation, and depolarization of the mitochondrial membrane. Further, inducing apoptosis of these complexes in A549 cancer cells and BEAS-2B normal cells also correlated with their anticancer selectivity, indicating the apoptosis mode of cell death in this system. In addition, these complexes could enter A549 cells via energy-dependent pathway and were able to impede the in vitro migration of A549 cells.

Electrochemical generation of 1-amino-pyrene-4,5,9,10 tetrol on the MWCNT surface for low potential electrocatalytic NADH oxidation

Electrochemical generation of 1-amino-pyrene-4,5,9,10 tetrol on the MWCNT surface for low potential electrocatalytic NADH oxidation

Insights into the binding of half-sandwich phosphino Ir(III) and Ru(II) complexes to deoxyribonucleic acid, albumin and apo-transferrin: Experimental and theoretical investigation

A group of cytotoxic half-sandwich iridium(III) (Ir(η5-Cp*)Cl2PPh2CH2OH (IrPOH)), (Ir(η5-Cp*)Cl2P(p-OCH3Ph)2CH2OH (IrMPOH)), and ruthenium(II) (Ru(η6-p-cymene)Cl2PPh2CH2OH (RuPOH), Ru(η6-p-cymene)Cl2P(p-OCH3Ph)2CH2OH (RuMPOH)) complexes with phosphine ligands exhibit the ability to (i) slow hydrolysis which is reversed by adding a high NaCl concentration; (ii) oxidation of NADH to NAD+; (iii) induction of cytotoxicity towards various cancer cell lines. Furthermore, we found that RuPOH and RuMPOH selectively inhibit the proliferation of skin cancer cells (WM266-4) while Ir(III) complexes were found to be moderate against prostate cancer cells (DU-145). Herein, to elucidate the cytotoxic effects, we investigated the interaction of these complexes with DNA and serum proteins by gel electrophoresis, fluorescence spectroscopy, and molecular docking studies. Fluorescence spectroscopic data (calf thymus DNA: CT-DNA titration), together with analysis of DNA fragmentation (gel electrophoresis) and molecular docking provided evidence for the multimodal interaction of Ir(III) and Ru(III) complexes with DNA with predominance of intercalation and minor groove binding. All examined complexes caused single-stranded cleavage of the sugar–phosphate backbone of plasmid DNA. The affinity of the complexes for apo-transferrin (apo-Tf) and human serum albumin (HSA) was evaluated by fluorescence emission spectroscopy to calculate the binding constants which suggested a tight and reversible binding. Moreover, ruthenium complexes can mimic the binding of iron compounds to specific biomolecules such as albumin and transferrin better than iridium complexes. In silico study indicate that complexes mostly bind to (i) apo-Tf with a preference for a single binding site and/or (ii) to dock within all the four predicted binding sites of HSA with the predominance of site I which include tryptophan residues of HSA. This class of ruthenium(II) and iridium(III) complexes has unusual features worthy of further exploration in the design of novel anticancer drugs.

Development and Application of an Electrochemical Sensor with 1,10-Phenanthroline-5,6-dione-Modified Electrode for the Detection of Escherichia coli in Water

The routine monitoring of bacterial populations is crucial for ensuring water quality and safeguarding public health. Thus, an electrochemical sensor based on a 1,10-phenanthroline-5,6-dione-modified electrode was developed and explored for the detection of E. coli. The modified electrode exhibited enhanced NADH oxidation ability at a low potential of 0.1 V, which effectively eliminated the interference from other redox compounds in bacteria. The sensitivity for NADH was 0.222 μA/μM, and the limit of detection was 0.0357 μM. Upon cell lysis, the intracellular NADH was released, and the concentration of E. coli was determined through establishing the relationship between the oxidation current signal and NADH concentration. The performance of the electrochemical sensor in the detection of NADH and E. coli suspensions was validated using the WST-8 colorimetric method. The blank recovery experiment in real water samples exhibited good accuracy, with recovery rates ranging from 89.12% to 93.26% and relative standard deviations of less than 10%. The proposed electrochemical sensor realized the detection of E. coli without the usage of biomarkers, which provides a promising approach for the broad-spectrum detection of microbial contents in complex water environments.

Abstract P1077: Transcription Factor Nfya Regulates Mitochondrial Metabolism And Cardiomyocyte Proliferation In The Fetal Heart

We previously identified a neonatal cardiomyocyte population that enters the cell-cycle following injury and disappears as the heart loses the ability to regenerate. These regenerative neonatal cardiomyocytes display a unique transcriptional program regulated by NFYA, a subunit of the trimeric complex Nuclear Transcription Factor Y (NFY) that functions as a pioneer factor to promote cell proliferation. To study whether NFYA is required for cardiomyocyte proliferation, we generated mice with cardiomyocyte-specific deletion of NFYA using alpha-MHC-Cre. NFYA KO mice died before birth; their hearts were smaller in ventricular volumes and developed a distinct cardiac noncompaction phenotype, with hyper trabeculation and reduced cardiomyocyte proliferation. Integration of single-nuclear RNA and ATAC sequencing with spatial transcriptomics identified different embryonic cardiomyocyte subtypes in wildtype and NFYA KO hearts, including those from atria, ventricles, trabeculae, and the conduction system. We found that NFYA deletion reduced the reservoir of immature regenerative cardiomyocytes in the fetal heart and increased the number of trabecular cardiomyocytes. Mechanistically, NFYA deletion reduced the expression of genes regulating oxidative phosphorylation metabolism. The defect in mitochondrial metabolism was further supported by decreased activity of NADH oxidation at mitochondrial membranes and reduced levels of TCA cycle metabolites in NFYA KO hearts. Using chromatin immunoprecipitation and reporter assays, we show that NFYA directly regulates the transcription of those mitochondrial metabolic genes as well as cell-cycle genes in cardiomyocytes, thus providing a transcriptional coupling between mitochondrial metabolism and cardiomyocyte proliferation. Our study identified a previously unrecognized transcriptional control mechanism of metabolic genes controlled by NFYA and highlights the importance of oxidative metabolism during fetal heart development and regeneration.

Cofactor Metabolic Engineering of Escherichia coli for Aerobic L-Malate Production with Lower CO2 Emissions.

Escherichia coli has been engineered for L-malate production via aerobic cultivation. However, the maximum yield obtained through this mode is inferior to that of anaerobic fermentation due to massive amounts of CO2 emissions. Here, we aim to address this issue by reducing CO2 emissions of recombinant E. coli during aerobic L-malate production. Our findings indicated that NADH oxidation and ATP-synthesis-related genes were down-regulated with 2 g/L of YE during aerobic cultivations of E. coli E23, as compared to 5 g/L of YE. Then, E23 was engineered via the knockout of nuoA and the introduction of the nonoxidative glycolysis (NOG) pathway, resulting in a reduction of NAD+ and ATP supplies. The results demonstrate that E23 (ΔnuoA, NOG) exhibited decreased CO2 emissions, and it produced 21.3 g/L of L-malate from glucose aerobically with the improved yield of 0.43 g/g. This study suggests that a restricted NAD+ and ATP supply can prompt E. coli to engage in incomplete oxidization of glucose, leading to the accumulation of metabolites instead of utilizing them in cellular respiration.

Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency.

Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.

The Coupling of Dihydroxybenzene Derivatives to MWCNT and Electrochemical NADH Oxidation

3,4 and 2,5- dihydroxybenzene compounds were separately and covalently coupled to the multiwalled carbon nanotube (MWCNT) deposited on the surface of glassy carbon electrode through electrochemical reduction of diazonium compound of benzylamine bearing Boc protecting group (tert-butyloxycarbonyl). Following the removal of the Boc group, an amide bond was formed between the amine-terminated surface and the acyl group of the dihydroxybenzene derivatives. The electrochemical properties of dihydroxybenzene-modified MWCNT and the influence of the sweep rate on the voltammogram were elucidated by cyclic voltammetry (CV). Electron transfer kinetics of modified MWCNT by dihydroxybenzene derivatives was also studied using Laviron’s theory. As evidenced by an improvement in the anodic peak current and a negative shift in the overpotential of NADH oxidation when compared to the bare MWCNT, 3,4 and 2,5- dihydroxybenzene modified MWCNT electrodes were shown to possess catalytic activity toward NADH oxidation.

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Optimization of energy production and central carbon metabolism in a non-respiring eukaryote

Most eukaryotes respire oxygen, using it to generate biomass and energy. However, a few organisms have lost the capacity to respire. Understanding how they manage biomass and energy production may illuminate the critical points at which respiration feeds into central carbon metabolism and explain possible routes to its optimization. Here, we use two related fission yeasts, Schizosaccharomyces pombe and Schizosaccharomyces japonicus, as a comparative model system. We show that although S.japonicus does not respire oxygen, unlike S.pombe, it is capable of efficient NADH oxidation, amino acid synthesis, and ATP generation. We probe possible optimization strategies through the use of stable isotope tracing metabolomics, mass isotopologue distribution analysis, genetics, and physiological experiments. S.japonicus appears to have optimized cytosolic NADH oxidation via glycerol-3-phosphate synthesis. It runs a fully bifurcated TCA pathway, sustaining amino acid production. Finally, we propose that it has optimized glycolysis to maintain high ATP/ADP ratio, in part by using the pentose phosphate pathway as a glycolytic shunt, reducing allosteric inhibition of glycolysis and supporting biomass generation. By comparing two related organisms with vastly different metabolic strategies, our work highlights the versatility and plasticity of central carbon metabolism in eukaryotes, illuminating critical adaptations supporting the preferential use of glycolysis over oxidative phosphorylation.

Dysregulated cellular redox status during hyperammonemia causes mitochondrial dysfunction and senescence by inhibiting sirtuin-mediated deacetylation.

Perturbed metabolism of ammonia, an endogenous cytotoxin, causes mitochondrial dysfunction, reduced NAD+ /NADH (redox) ratio, and postmitotic senescence. Sirtuins are NAD+ -dependent deacetylases that delay senescence. In multiomics analyses, NAD metabolism and sirtuin pathways are enriched during hyperammonemia. Consistently, NAD+ -dependent Sirtuin3 (Sirt3) expression and deacetylase activity were decreased, and protein acetylation was increased in human and murine skeletal muscle/myotubes. Global acetylomics and subcellular fractions from myotubes showed hyperammonemia-induced hyperacetylation of cellular signaling and mitochondrial proteins. We dissected the mechanisms and consequences of hyperammonemia-induced NAD metabolism by complementary genetic and chemical approaches. Hyperammonemia inhibited electron transport chain components, specifically complex I that oxidizes NADH to NAD+ , that resulted in lower redox ratio. Ammonia also caused mitochondrial oxidative dysfunction, lower mitochondrial NAD+ -sensor Sirt3, protein hyperacetylation, and postmitotic senescence. Mitochondrial-targeted Lactobacillus brevis NADH oxidase (MitoLbNOX), but not NAD+ precursor nicotinamide riboside, reversed ammonia-induced oxidative dysfunction, electron transport chain supercomplex disassembly, lower ATP and NAD+ content, protein hyperacetylation, Sirt3 dysfunction and postmitotic senescence in myotubes. Even though Sirt3 overexpression reversed ammonia-induced hyperacetylation, lower redox status or mitochondrial oxidative dysfunction were not reversed. These data show that acetylation is a consequence of, but is not the mechanism of, lower redox status or oxidative dysfunction during hyperammonemia. Targeting NADH oxidation is a potential approach to reverse and potentially prevent ammonia-induced postmitotic senescence in skeletal muscle. Since dysregulated ammonia metabolism occurs with aging, and NAD+ biosynthesis is reduced in sarcopenia, our studies provide a biochemical basis for cellular senescence and have relevance in multiple tissues.

ECSIT is essential for RANKL-induced stimulation of mitochondria in osteoclasts and a target for the anti-osteoclastogenic effects of estrogens.

Estrogens inhibit bone resorption and preserve bone mass, at least in part, via direct effects on osteoclasts. The binding of RANKL, the critical cytokine for osteoclast differentiation, to its receptor in osteoclast precursor cells of the monocyte lineage recruits the adaptor protein TRAF6 and activates multiple signaling pathways. Early effects of RANKL include stimulation of mitochondria. 17β-estradiol (E2) prevents the effects of RANKL on mitochondria and promotes mitochondria mediated apoptotic cell death. However, the molecular mechanisms responsible for the actions of RANKL and estrogens on mitochondria remain unknown. Evolutionarily Conserved Signaling Intermediate in Toll Pathway (ECSIT) is a complex I-associated protein that regulates immune responses in macrophages following the engagement of Toll-like receptors, which also recruit TRAF6. Here, we examined whether ECSIT could be implicated in the rapid effects of RANKL and E2 on osteoclast progenitors. Bone marrow-derived macrophages (BMMs) from C57BL/6 mice were cultured with RANKL (30 ng/ml) with or without E2 (10-8 M). ECSIT-TRAF6 interaction was evaluated by co-immunoprecipitation and ECSIT levels in mitochondria and cytosolic fractions by Western blot. ShRNA lentivirus particles were used to knockdown ECSIT. Osteoclasts were enumerated after tartrate-resistant acid phosphatase staining. Oxygen consumption and extracellular acidification rates were measured with Seahorse XFe96 Analyzer. ATP, lactate, and NAD/NADH were measured with commercial assay kits. NADH oxidation to NAD was used to evaluate Complex I activity. Total and mitochondrial ROS, and mitochondrial membrane potential were measured with H2DCFDA, MitoSOX, and TMRM probes, respectively. Degradation of DEVD-AFC was used to measure Caspase-3 activity. We found that RANKL promoted ECSIT-TRAF6 interaction and increased the levels of ECSIT in mitochondria. E2 abrogated these effects of RANKL. Silencing of ECSIT decreased osteoclast differentiation and abrogated the inhibitory effects of E2 on osteoclastogenesis. Loss of ECSIT decreased complex I activity, oxygen consumption, NAD+/NADH redox ratio, and ATP production and increased mitochondrial ROS. In the absence of ECSIT, the stimulatory actions of RANKL on complex I activity and all other markers of oxidative phosphorylation, as well as their inhibition by E2, were prevented. Instead, RANKL stimulated apoptosis of osteoclast progenitors. These findings suggest that dysregulated mitochondria cause a switch in RANKL signaling from pro-survival to pro-apoptotic. In addition, our results indicate that ECSIT represents a central node for the early effects of RANKL on mitochondria and that inhibition of ECSIT-mediated mitochondria stimulation might contribute to the bone protective actions of estrogens.

Two separate pathways underlie NADH and succinate oxidation in swine heart mitochondria: Kinetic evidence on the mobile electron carriers

We have investigated NADH and succinate aerobic oxidation in frozen and thawed swine heart mitochondria. Simultaneous oxidation of NADH and succinate showed complete additivity under a variety of experimental conditions, suggesting that the electron fluxes originating from NADH and succinate are completely independent and do not mix at the level of the so-called mobile diffusible components. We ascribe the results to mixing of the fluxes at the level of cytochrome c in bovine mitochondria: the Complex IV flux control coefficient in NADH oxidation was high in swine mitochondria but very low in bovine mitochondria, suggesting a stronger interaction of cytochrome c with the supercomplex in the former. This was not the case in succinate oxidation, in which Complex IV exerted little control also in swine mitochondria. We interpret the data in swine mitochondria as restriction of the NADH flux by channelling within the I-III2-IV supercomplex, whereas the flux from succinate shows pool mixing for both Coenzyme Q and probably cytochrome c. The difference between the two types of mitochondria may be ascribed to different lipid composition affecting the cytochrome c binding properties, as suggested by breaks in Arrhenius plots of Complex IV activity occurring at higher temperatures in bovine mitochondria.

Specific Features of Mitochondrial Dysfunction under Conditions of Ferroptosis Induced by t-Butylhydroperoxide and Iron: Protective Role of the Inhibitors of Lipid Peroxidation and Mitochondrial Permeability Transition Pore Opening

Recent studies have indicated the critical importance of mitochondria in the induction and progression of ferroptosis. There is evidence indicating that tert-butyl hydroperoxide (TBH), a lipid-soluble organic peroxide, is capable of inducing ferroptosis-type cell death. We investigated the effect of TBH on the induction of nonspecific membrane permeability measured by mitochondrial swelling and on oxidative phosphorylation and NADH oxidation assessed by NADH fluo rescence. TBH and iron, as well as their combinations, induced, with a respective decrease in the lag phase, the swelling of mitochondria, inhibited oxidative phosphorylation and stimulated NADH oxidation. The lipid radical scavenger butylhydroxytoluene (BHT), the inhibitor of mitochondrial phospholipase iPLA2γ bromoenol lactone (BEL), and the inhibitor of the mitochondrial permeability transition pore (MPTP) opening cyclosporine A (CsA) were equally effective in protecting these mitochondrial functions. The radical-trapping antioxidant ferrostatin-1, a known indicator of ferroptotic alteration, restricted the swelling but was less effective than BHT. ADP and oligomycin significantly decelerated iron- and TBH-induced swelling, confirming the involvement of MPTP opening in mitochondrial dysfunction. Thus, our data showed the participation of phospholipase activation, lipid peroxidation, and the MPTP opening in the mitochondria-dependent ferroptosis. Presumably, their involvement took place at different stages of membrane damage initiated by ferroptotic stimuli.

Phosphorylation of GntR reduces Streptococcus suis oxidative stress resistance and virulence by inhibiting NADH oxidase transcription.

GntR transcription factor of Streptococcus suis serotype 2 (SS2) is a potential substrate protein of STK, but the regulation mechanisms of GntR phosphorylation are still unclear. This study confirmed that STK phosphorylated GntR in vivo, and in vitro phosphorylation experiments showed that STK phosphorylated GntR at Ser-41. The phosphomimetic strain (GntR-S41E) had significantly reduced lethality in mice and reduced bacterial load in the blood, lung, liver, spleen, and brain of infected mice compared to wild-type (WT) SS2. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated that the promoter of nox was bound by GntR. The phosphomimetic protein GntR-S41E cannot bind to the promoter of nox, and the nox transcription levels were significantly reduced in the GntR-S41E mutant compared to WT SS2. The virulence in mice and the ability to resist oxidative stress of the GntR-S41E strain were restored by complementing transcript levels of nox. NOX is an NADH oxidase that catalyzes the oxidation of NADH to NAD+ with the reduction of oxygen to water. We found that NADH is likely accumulated under oxidative stress in the GntR-S41E strain, and higher NADH levels resulted in increased amplified ROS killing. In total, we report GntR phosphorylation could inhibit the transcription of nox, which impaired the ability of SS2 to resist oxidative stress and virulence.